Ocimum kilimandscharicum 4CL11 negatively regulates adventitious root development via accumulation of flavonoid glycosides.

4-Coumarate-CoA Ligase (4CL) is an important enzyme in the phenylpropanoid biosynthesis pathway. Multiple 4CLs are identified in Ocimum species; however, their in planta functions remain enigmatic. In this study, we independently overexpressed three Ok4CL isoforms from Ocimum kilimandscharicum (Ok4CL7, -11, and -15) in Nicotiana benthamiana. Interestingly, Ok4CL11 overexpression (OE) caused a rootless or reduced root growth phenotype, whereas overexpression of Ok4CL15 produced normal adventitious root (AR) growth. Ok4CL11 overexpression in N. benthamiana resulted in upregulation of genes involved in flavonoid biosynthesis and associated glycosyltransferases accompanied by accumulation of specific flavonoid-glycosides (kaempferol-3-rhamnoside, kaempferol-3,7-O-bis-alpha-l-rhamnoside [K3,7R], and quercetin-3-O-rutinoside) that possibly reduced auxin levels in plants, and such effects were not seen for Ok4CL7 and -15. Docking analysis suggested that auxin transporters (PINs/LAXs) have higher binding affinity to these specific flavonoid-glycosides, and thus could disrupt auxin transport/signaling, which cumulatively resulted in a rootless phenotype. Reduced auxin levels, increased K3,7R in the middle and basal stem sections, and grafting experiments (intra and inter-species) indicated a disruption of auxin transport by K3,7R and its negative effect on AR development. Supplementation of flavonoids and the specific glycosides accumulated by Ok4CL11-OE to the wild-type N. benthamiana explants delayed the AR emergence and also inhibited AR growth. While overexpression of all three Ok4CLs increased lignin accumulation, flavonoids, and their specific glycosides were accumulated only in Ok4CL11-OE lines. In summary, our study reveals unique indirect function of Ok4CL11 to increase specific flavonoids and their glycosides, which are negative regulators of root growth, likely involved in inhibition of auxin transport and signaling.

Engineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis.

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

MicroRNA-129-5p-regulated microglial expression of the surface receptor CD200R1 controls neuroinflammation.

CD200R1 is an inhibitory surface receptor expressed in microglia and blood macrophages. Microglial CD200R1 is known to control neuroinflammation by keeping the microglia in resting state, and therefore, tight regulation of its expression is important. CCAAT/enhancer-binding protein ß (CEBPß) is the known regulator of CD200R1 transcription. In the present study, our specific intention was to find a possible posttranscriptional regulatory mechanism of CD200R1 expression. Here we investigated a novel regulatory mechanism of CD200R1 expression following exposure to an environmental stressor, arsenic, combining in silico analysis, in vitro, and in vivo experiments, as well as validation in human samples. The in silico analysis and in vitro studies with primary neonatal microglia and BV2 microglia revealed that arsenic demethylates the promoter of a microRNA, miR-129-5p, thereby increasing its expression, which subsequently represses CD200R1 by binding to its 3'-untranslated region and shuttling the CD200R1 mRNA to the cytoplasmic-processing body in mouse microglia. The role of miR-129-5p was further validated in BALB/c mouse by stereotaxically injecting anti-miR-129. We found that anti-miR-129 reversed the expression of CD200R1, as well as levels of inflammatory molecules IL-6 and TNF-α. Experiments with a CD200R1 siRNA-induced loss-of-function mouse model confirmed an miR-129-5p→CD200R1→IL-6/TNF-α signaling axis. These main findings were replicated in a human cell line and validated in human samples. Taken together, our study revealed miR-129-5p as a novel posttranscriptional regulator of CD200R1 expression with potential implications in neuroinflammation and related complications.

Genomic Determinants of Entomopathogenic Fungi and Their Involvement in Pathogenesis.

Entomopathogenic fungi offer an effective and eco-friendly alternative to curb insect populations in biocontrol strategy. The evolutionary history of selected entomopathogenic fungi indicates their ancestral relationship with plant endophytes. During this host shifting, entomopathogenic fungi must have acquired multiple mechanisms, including a combination of various biomolecules that make them distinguishable from other fungi. In this review, we focus on understanding various biochemical and molecular mechanisms involved in entomopathogenesis. In particular, we attempt to explain the indispensable role of enlarged gene families of various virulent factors, viz. chitinases, proteases, lipases, specialized metabolites, and cytochrome P450, in entomopathogenesis. Our analysis suggests that entomopathogenic fungi recruit a different set of gene products during the progression of pathogenesis. Knowledge of these bio-molecular interactions between fungi and insect hosts will allow researchers to execute pointed efforts towards the development of improved entomopathogenic fungal strains.

Functional Diversity of the Lepidopteran ATP-Binding Cassette Transporters.

The ATP-binding cassette (ABC) transporter gene family is ubiquitous in the living world. ABC proteins bind and hydrolyze ATP to transport a myriad of molecules across various lipid-containing membrane systems. They have been studied well in plants for transport of a variety of compounds and particularly, in vertebrates due to their direct involvement in resistance mechanisms against several toxic molecules/metabolites. ABC transporters in insects are found within large multigene families involved in the efflux of chemical insecticides and toxic/undesired metabolites originating from food and endogenous metabolism. This review deals with ABC transporter subfamilies of few agronomically important Lepidopteran pests. The transcriptional dynamics and regulation of ABC transporters during insect development emphasizes their functional diversity against insecticides, Cry toxins, and plant specialized metabolites. To generate insights about molecular function and physiological roles of ABCs, functional and structural characterization is necessary. Also, expansion and divergence of ABC transporter gene subfamilies in Lepidopteran insects needs more systematic investigation. We anticipate that newer methods of insect control in agriculture can benefit from an understanding of ABC transporter interactions with a vast range of natural specialized molecules and synthetic compounds.

2-oxoglutarate-dependent dioxygenases drive expansion of steroidal alkaloid structural diversity in the genus Solanum.

Solanum steroidal glycoalkaloids (SGAs) are renowned defence metabolites exhibiting spectacular structural diversity. Genes and enzymes generating the SGA precursor pathway, SGA scaffold and glycosylated forms have been largely identified. Yet, the majority of downstream metabolic steps creating the vast repertoire of SGAs remain untapped. Here, we discovered that members of the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (2-ODD) family play a prominent role in SGA metabolism, carrying out three distinct backbone-modifying oxidative steps in addition to the three formerly reported pathway reactions. The GLYCOALKALOID METABOLISM34 (GAME34) enzyme catalyses the conversion of core SGAs to habrochaitosides in wild tomato S. habrochaites. Cultivated tomato plants overexpressing GAME34 ectopically accumulate habrochaitosides. These habrochaitoside enriched plants extracts potently inhibit Puccinia spp. spore germination, a significant Solanaceae crops fungal pathogen. Another 2-ODD enzyme, GAME33, acts as a desaturase (via hydroxylation and E/F ring rearrangement) forming unique, yet unreported SGAs. Conversion of bitter α-tomatine to ripe fruit, nonbitter SGAs (e.g. esculeoside A) requires two hydroxylations; while the known GAME31 2-ODD enzyme catalyses hydroxytomatine formation, we find that GAME40 catalyses the penultimate step in the pathway and generates acetoxy-hydroxytomatine towards esculeosides accumulation. Our results highlight the significant contribution of 2-ODD enzymes to the remarkable structural diversity found in plant steroidal specialized metabolism.

Steroidal alkaloids defence metabolism and plant growth are modulated by the joint action of gibberellin and jasmonate signalling.

Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.

A glance at the chemodiversity of Ocimum species: Trends, implications, and strategies for the quality and yield improvement of essential oil.

Ocimum species represent commercially important medicinal and aromatic plants. The essential oil biosynthesized by Ocimum species is enriched with specialized metabolites specifically, terpenoids and phenylpropanoids. Interestingly, various Ocimum species are known to exhibit diverse chemical profiles, and this chemical diversity has been at the center of many studies to identify commercially important chemotypes. Here, we present various chemotypes from the Ocimum species and emphasize trends, implications, and strategies for the quality and yield improvement of essential oil. Globally, many Ocimum species have been analyzed for their essential oil composition in over 50 countries. Asia represents the highest number of chemotypes, followed by Africa, South America, and Europe. Ocimum basilicum L. has been the most widespread and well-studied species, followed by O. gratissimum L., O. tenuiflorum L., O. canum Sims, O. americanum and O. kilimandscharicum Gürke. Moreover, various molecular reasons, benefits, adverse health effects and mechanisms behind this vast chemodiversity have been discussed. Different strategies of plant breeding, metabolic engineering, transgenic, and tissue-culture, along with anatomical modifications, are surveyed to enhance specific chemotypic profiles and essential oil yield in numerous Ocimum species. Consequently, chemical characterization of the essential oil obtained from Ocimum species has become indispensable for its proper utilization. The present chemodiversity knowledge from Ocimum species will help to exploit various applications in the industrial, agriculture, biopharmaceutical, and food sectors. Supplementary Information: The online version contains supplementary material available at 10.1007/s11101-021-09767-z.

PINIR: a comprehensive information resource for Pin-II type protease inhibitors.

BACKGROUND: Serine protease inhibitors belonging to the Potato type-II Inhibitor family Protease Inhibitors (Pin-II type PIs) are essential plant defense molecules. They are characterized by multiple inhibitory repeat domains, conserved disulfide bond pattern, and a tripeptide reactive center loop. These features of Pin-II type PIs make them potential molecules for protein engineering and designing inhibitors for agricultural and therapeutic applications. However, the diversity in these PIs remains unexplored due to the lack of annotated protein sequences and their functional attributes in the available databases. RESULTS: We have developed a database, PINIR (Pin-II type PIs Information Resource), by systematic collection and manual annotation of 415 Pin-II type PI protein sequences. For each PI, the number and position for signature sequences are specified: 695 domains, 75 linkers, 63 reactive center loops, and 10 disulfide bond patterns are identified and mapped. Database analysis revealed novel subcategories of PIs, species-correlated occurrence of inhibitory domains, reactive center loops, and disulfide bond patterns. By analyzing linker regions, we predict that alternative processing at linker regions could generate PI variants in the Solanaceae family. CONCLUSION: PINIR ( https://pinir.ncl.res.in ) provides a web interface for browsing and analyzing the protein sequences of Pin-II type PIs. Information about signature sequences, spatio-temporal expression, biochemical properties, gene sequences, and literature references are provided. Analysis of PINIR depicts conserved species-specific features of Pin-II type PI protein sequences. Diversity in the sequence of inhibitory domains and reactive loops directs potential applications to engineer Pin-II type PIs. The PINIR database will serve as a comprehensive information resource for further research into Pin-II type PIs.

Influence of domestication on specialized metabolic pathways in fruit crops.

MAIN CONCLUSION: During the process of plant domestication, the selection and traditional breeding for desired characters such as flavor, juiciness and nutritional value of fruits, probably have resulted in gain or loss of specialized metabolites contributing to these traits. Their appearance in fruits is likely due to the acquisition of novel and specialized metabolic pathways and their regulation, driven by systematic molecular evolutionary events facilitated by traditional breeding. Plants change their armory of specialized metabolism to adapt and survive in diverse ecosystems. This may occur through molecular evolutionary events, such as single nucleotide polymorphism, gene duplication and transposition, leading to convergent or divergent evolution of biosynthetic pathways producing such specialized metabolites. Breeding and selection for improved specific and desired traits (fruit size, color, taste, flavor, etc.) in fruit crops through conventional breeding approaches may further alter content and profile of specialized metabolites. Biosynthetic routes of these metabolites have been studied in various plants. Here, we explore the influence of plant domestication and breeding processes on the selection of biosynthetic pathways of favorable specialized metabolites in fruit crops. An orderly clustered arrangement of genes associated with their production is observed in many fruit crops. We further analyzed selection-based acquisition of specialized metabolic pathways comparing first the metabolic profiles and genes involved in their biosynthesis, followed by the genomic organization of such genes between wild and domesticated horticultural crops. Domestication of crop plants favored the acquisition and retention of metabolic pathways that enhanced the fruit value while eliminated those which produced toxic or unfavorable metabolites. Interestingly, unintentional reorganization of complex metabolic pathways by selection and traditional breeding processes has endowed us with flavorful, juicy and nutritionally rich fruits.

Theaflavin-Containing Black Tea Extract: A Potential DNA Methyltransferase Inhibitor in Human Colon Cancer Cells and Ehrlich Ascites Carcinoma-Induced Solid Tumors in Mice.

Tea is the most popularly consumed beverage in the world. Theaflavin and thearubigins are the key bioactive compounds of black tea that have anticarcinogenic properties as reported in several studies. However, the epigenetic potential of these compounds has not yet been explored. DNA methyltransferase (DNMT) enzymes induce methylation of DNA at cytosine residues and play a significant role in epigenetic regulation and cancer therapy. The present study has explored the role of black tea as a DNMT inhibitor in the prevention of cancer. Herein, the effect of theaflavin has been studied in colon cancer cell line (HCT-116) and EAC-induced solid tumors in mice. It was found that theaflavin prevented cell proliferation and inhibited tumor progression as well. In silico study showed that theaflavin interacted with DNMT1 and DNMT3a enzymes and blocked their activity. Theaflavin also decreased DNMT activity In Vitro and In Vivo as evident from the DNMT activity assay. Results of immunohistochemistry revealed that theaflavin reduced DNMT expression in the tumors of mice. Taken together, our findings showed that theaflavin has a potential role as a DNMT inhibitor in HCT-116 cell line and EAC induced solid tumors in mice.

Diversity in Chemical Structures and Biological Properties of Plant Alkaloids.

Phytochemicals belonging to the group of alkaloids are signature specialized metabolites endowed with countless biological activities. Plants are armored with these naturally produced nitrogenous compounds to combat numerous challenging environmental stress conditions. Traditional and modern healthcare systems have harnessed the potential of these organic compounds for the treatment of many ailments. Various chemical entities (functional groups) attached to the central moiety are responsible for their diverse range of biological properties. The development of the characterization of these plant metabolites and the enzymes involved in their biosynthesis is of an utmost priority to deliver enhanced advantages in terms of biological properties and productivity. Further, the incorporation of whole/partial metabolic pathways in the heterologous system and/or the overexpression of biosynthetic steps in homologous systems have both become alternative and lucrative methods over chemical synthesis in recent times. Moreover, in-depth research on alkaloid biosynthetic pathways has revealed numerous chemical modifications that occur during alkaloidal conversions. These chemical reactions involve glycosylation, acylation, reduction, oxidation, and methylation steps, and they are usually responsible for conferring the biological activities possessed by alkaloids. In this review, we aim to discuss the alkaloidal group of plant specialized metabolites and their brief classification covering major categories. We also emphasize the diversity in the basic structures of plant alkaloids arising through enzymatically catalyzed structural modifications in certain plant species, as well as their emerging diverse biological activities. The role of alkaloids in plant defense and their mechanisms of action are also briefly discussed. Moreover, the commercial utilization of plant alkaloids in the marketplace displaying various applications has been enumerated.

Integrated Transcriptomic and Proteomic Analyses Suggest the Participation of Endogenous Protease Inhibitors in the Regulation of Protease Gene Expression in Helicoverpa armigera.

Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of multi-domain recombinant Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant CanPI-7 at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression on CanPI-7 feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h). Further, coexpression of H. armigera endogenous PIs with several digestive protease genes were apparent. In addition to the differential expression of endogenous H. armigera PIs, we also observed a distinct novel isoform of endogenous PI in CanPI-7 fed H. armigera larvae. Based on present and earlier studies, we propose potential mechanism of protease regulation in H. armigera and subsequent adaptation strategy to cope with anti-nutritional components of plants.

Semirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity.

Uridine diphosphate-dependent glycosyltransferases (UGTs) catalyze the transfer of a diversity of sugars to several acceptor molecules and often exhibit distinct substrate specificity. Modulation of glycosyltransferases for increased catalytic activity and altered substrate or product specificity are the key manipulations for the biotechnological use of glycosyltransferases in various biosynthetic processes. Here, we have engineered the binding pocket of three previously characterized Vitis vinifera glycosyltransferases, UGT88F12, UGT72B27 and UGT92G6, by structure-guided in silico mutagenesis to facilitate the interactions of active site residues with flavonol glucosides and thus modify substrate specificity and activity. Site-directed mutagenesis at selected sites, followed with liquid chromatography-mass spectrometry based activity assays, exhibited that mutant UGTs were altered in product selectivity and activity as compared to the wild-type enzymes. Mutant UGTs produced larger amounts of flavonol di-monosaccharide glucosides, which imply that the mutations led to structural changes that increased the volume of the binding pocket to accommodate a larger substrate and to release larger products at ease. Mutants showed increased activity and modified product specificity. Thus, structure-based systematic mutations of the amino acid residues in the binding pocket can be explored for the generation of engineered UGTs for diverse biotechnological applications.

High-throughput metabolomic and transcriptomic analyses vet the potential route of cerpegin biosynthesis in two varieties of Ceropegia bulbosa Roxb.

MAIN CONCLUSION: Exploration with high-throughput transcriptomics and metabolomics of two varieties of Ceropegia bulbosa identifies candidate genes, crucial metabolites and a potential cerpegin biosynthetic pathway. Ceropegia bulbosa is an important medicinal plant, used in the treatment of various ailments including diarrhea, dysentery, and syphilis. This is primarily attributed to the presence of pharmaceutically active secondary metabolites, especially cerpegin. As this plant belongs to an endemic threatened category, genomic resources are not available hampering exploration on the molecular basis of cerpegin accumulation till now. Therefore, we undertook high-throughput metabolomic and transcriptomic analyses using different tissues from two varieties namely, C. bulbosa var. bulbosa and C. bulbosa var. lushii. Metabolomic analysis revealed spatial and differential accumulation of various metabolites. We chemically synthesized and characterized the cerpegin and its derivatives by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Importantly, these comparisons suggested the presence of cerpegin and 5-allyl cerpegin in all C. bulbosa tissues. Further, de novo transcriptome analysis indicated the presence of significant transcripts for secondary metabolic pathways through the Kyoto encyclopedia of genes and genomes database. Tissue-specific profiling of transcripts and metabolites showed a significant correlation, suggesting the intricate mechanism of cerpegin biosynthesis. The expression of potential candidate genes from the proposed cerpegin biosynthetic pathway was further validated by qRT-PCR and NanoString nCounter. Overall, our findings propose a potential route of cerpegin biosynthesis. Identified transcripts and metabolites have built a foundation as new molecular resources that could facilitate future research on biosynthesis, regulation, and engineering of cerpegin or other important metabolites in such non-model plants.

Terpene profiling, transcriptome analysis and characterization of cis-ß-terpineol synthase from Ocimum.

Ocimum species produces a varied mix of different metabolites that imparts immense medicinal properties. To explore this chemo-diversity, we initially carried out metabolite profiling of different tissues of five Ocimum species and identified the major terpenes. This analysis broadly classified these five Ocimum species into two distinct chemotypes namely, phenylpropanoid-rich and terpene-rich. In particular, ß-caryophyllene, myrcene, limonene, camphor, borneol and selinene were major terpenes present in these Ocimum species. Subsequently, transcriptomic analysis of pooled RNA samples from different tissues of Ocimum gratissimum, O. tenuiflorum and O. kilimandscharicum identified 38 unique transcripts of terpene synthase (TPS) gene family. Full-length gene cloning, followed by sequencing and phylogenetic analysis of three TPS transcripts were carried out along with their expression in various tissues. Terpenoid metabolite and expression profiling of candidate TPS genes in various tissues of Ocimum species revealed spatial variances. Further, putative TPS contig 19414 (TPS1) was selected to corroborate its role in terpene biosynthesis. Agrobacterium-mediated transient over-expression assay of TPS1 in the leaves of O. kilimandscharicum and subsequent metabolic and gene expression analyses indicated it as a cis-ß-terpineol synthase. Overall, present study provided deeper understanding of terpene diversity in Ocimum species and might help in the enhancement of their terpene content through advanced biotechnological approaches.

Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases.

Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.

Structural, functional and evolutionary diversity of 4-coumarate-CoA ligase in plants.

MAIN CONCLUSIONS: The 4-coumarate-CoA ligases (4CL) contribute in channelizing flux of different phenylpropanoid biosynthetic pathways. Expression of 4CL is optimized at developmental stages and in response to environmental triggers such as biotic and abiotic stresses. The enzyme is valuable in metabolic pathway engineering for curcuminoids, resveratrol, biofuel production and nutritional improvement. Vigorous analysis of regulation at functional and expression level is obligatory to attain efficient commercial production of candidate metabolites using 4CL. Phenylpropanoid pathway provides precursors for numerous secondary metabolites in plants. In this pathway, 4-coumarate-CoA ligase (EC 6.2.1.12, 4CL) is the main branch point enzyme which generates activated thioesters. Being the last enzyme of three shared common steps in general phenylpropanoid pathway, it contributes to channelize precursors for different phenylpropanoids. In plants, 4CL enzymes are present in multiple isoforms and encoded by small gene family. It belongs to adenylate-forming enzyme family and catalyzes the reaction that converts hydroxy or methoxy cinnamic acid derivatives to corresponding thioesters. These thioesters are further utilized for biosynthesis of phenylpropanoids, which are known for having numerous nutritional and medicinal applications. In addition, the 4CL enzymes have been characterized from various plants for their role in plant physiology or in biotic and abiotic stresses. Furthermore, specific isoforms are differentially regulated upon exposure to diverse stimuli leading to flux diversion toward the particular metabolite biosynthesis. Evolutionary studies showed that 4CL separately evolved after monocot and dicot segregation. Here, we provide a comprehensive review on 4CL, which includes evolution, function, gene/protein structure, role in metabolite biosynthesis and cellular partition, and their regulation. Based on the available data, we have explored the scope for pathway engineering by utilizing 4CL enzymes.

WRKY1 acts as a key component improving resistance against Alternaria solani in wild tomato, Solanum arcanum Peralta.

Early blight (EB), caused by Alternaria solani, is a major threat to global tomato production. In comparison with cultivated tomato (Solanum lycopersicum), a wild relative, S. arcanum exhibits strong resistance against EB. However, molecular cascades operating during EB resistance in wild or cultivated tomato plants are largely obscure. Here, we provide novel insight into spatio-temporal molecular events in S. arcanum against A. solani. Transcriptome and co-expression analysis presented 33-WRKYs as promising candidates of which 12 SaWRKYs displayed differential expression patterns in resistant and susceptible accessions during EB disease progression. Among these, SaWRKY1 exhibited induced expression with significant modulation in xyloglucan endotrans hydrolase 5 (XTH5) and MYB2 expressions that correlated with the disease phenotypes. Electro-mobility shift assay confirmed physical interaction of recombinant SaWRKY1 to SaXTH5 and SaMYB2 promoters. Comparative WRKY1 promoter analysis between resistant and susceptible plants revealed the presence of crucial motifs for defence mechanism exclusively in resistant accession. Additionally, many defence-related genes displayed significant expression variations in both the accessions. Further, WRKY1 overexpressing transgenic plants exhibited higher levels of EB resistance while RNAi silencing lines had increased susceptibility to A. solani with altered expression of XTH5 and MYB2. Overall, these findings demonstrate the positive influence of WRKY1 in improving EB resistance in wild tomato and this could be further utilized as a potential target through genetic engineering to augment protection against A. solani in crop plants.